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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/61—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving triglycerides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase

Definitions

• the present invention relates to a reagent system and assay methodology for the quantitative estimation of triglycerides in biological fluids.
• Triglycerides which are triesters of fatty acids with glycerol, have very significant importance to centuries, in that they provide potent storage forms of energy in human biochemistry. About 18% of the total body weight in man is composed of adipose tissue, which in turn is comprised of about 90% (dry weight) of triglycerides; and it is the catabolism of these triglycerides which represents the major source of all body energy available to man, with the exception of glucose metabolism.
• triglycerides As triglycerides represent energy storage forms, they are mobilized and circulated in the bloodstream associated with protein carrier molecules known as lipoproteins.
• hypertriglyceridemia has also been associated with certain types of liver disease, diabetes mellitus, pancreatitis and glycogen storage disease.
• the present invention greatly advances the state of triglyceride assays, as well as simplifies the technique involved, thus making this important and critical preventative test available to greater segments of the population; and the higher availability of a reliable triglyceride assay, in turn, provides for many persons a healthier and fuller and longer life.
• Triglycerides which are members of a general class of compounds known as lipids or fats (other major constituents of this class of compounds being phospholipids, cholesterol, and cholester esters) have had various assays proposed and attempted through the years.
• the analyst would then try to determine, as best he could, by other procedures, the serum content of phospholipids, cholesterol, and cholesterol esters.
• Triglyceride content would then be estimated by subtracting the thus-estimated weight of phospholipids, cholesterol, and cholesterol esters from the relatively inaccurate weight of the total lipid measured, according to the following formula:
• Triglyceride Total lipids-[phospholipids+cholesterol+1.67 cholesterol esters] where the 1.67 is a factor based on the average molecular weight of the cholesterol esters.
• Van Handel and Zilversmit 3 attempted a variation in which serum lipids were extracted with a chloroform-methanol mixture, and the phospholipids removed from the organic solvent by treatment with Doucil (a zeolite). The triglycerides remaining in the solvent were then hydrolyzed, and the liberated glycerol was oxidized to formaldehyde with periodic acid according to the procedure of Lambert and Neish 4 . The formaldehyde so formed was then reacted with chromotropic acid to form a violet chromophore which was then spectrophotometrically quantitated.
• Doucil a zeolite
• Carlson and Wadstrom 5 then attempted a hydrolytic technique, similar to that of Van Handel and Zilversmit except that silicic acid was used to remove phospholipids. In addition, an extra-extraction step was included to remove fatty acids from the organic phase.
• the mixture was then extracted with a portion of petroleum ether to remove fatty acids. Then the glycerol was extracted with sulfuric acid, and oxidized to formaldehyde with sodium periodate. The excess periodate was then destroyed by the attition of sodium arsenite, and the formaldehyde would then be reacted with chromotropic acid to give a violet chromophore which was spectrophotometrically read at a wave length of 570 namometers.
• Lofland 6 proposed a hydrolytic procedure for the determination of triglycerides, in which the latter portion of the assay was carried out by automated means.
• the serum lipids were first extracted with isopropanol, and the phospholipids removed by treatment with zeolite.
• the extracts were then saponified by alkali; and then the reaction of glycerol with periodate to form formaldehyde, and subsequent estimation of the formaldehyde with chromotropic acid, was done by automated means.
• Kessler and Lederer 7 proposed a modification of the Lofland method, in which serum is extracted with isopropanol and the phospholipids are removed with zeolite; in this method, the subsequent saponification, oxidation to formaldehyde, and colorimetric estimation of formaldehyde, were all done by automated means. Kessler and Lederer, however, found that considerable amounts of glucose could be carried into the organic phase during the extraction process, and when subjected to periodate would give false positive reactions.
• the zeolite was combined with small quantities of copper sulfate and calcium hydroxide to destroy the glucose present. Additionally, the formaldehyde was estimated by reaction with a beta-diketone, and an amine, to form a lutidine-type compound which would then be estimated by fluoresence techniques.
• each molecule of glycerol would in turn run through the reaction sequence and convert one molecule of NADH to NAD with a corresponding loss of absorbance when measured at 340 nanometers in a spectrophotometer.
• This enzyme which is a common constituent of serum and which may vary markedly between patients, had the effect of catalyzing the conversion of ATP to ADP; and this in turn would cause the phosphoenol pyruvate to convert to pyruvate, etc., culminating in a false overestimation of glycerol.
• this blank correction factor necessitated an increase in the time necessary to accomplish the procedure as well as almost doubling the needed amount of an already expensive reagent system.
• the system measured color loss; and thus for a specimen which contained no triglycerides, one would obtain a very large number for the spectrophotometric absorbance, indicating properly that the NADH which absorbs at 340 nanometers was still present.
• the color loss would be small; and thus in the low range the assay is liable to inaccuracy due to the type of error usually involved in a calculation dependent upon a difference of very large numbers. This difficulty would ultimately manifest itself as a lack of precision or reproducibility at low levels.
• a second problem common to systems which measure the NADH system at 340 nanometers is that it is a well known fact that lipemia interferes to a much greater degree at low wavelengths such as at 340 nanometers, as opposed, for example, to measurements at 600-700 nanometers. This interference expresses itself as increased absorbance readings, further compounding the inaccuracy problems cited above.
• the third common problem attendant with this type system is that the chromophore itself is comparatively insensitive, that is, each interconversion of NADH/NAD produces a small absorbance change, thus limiting the analyst in his ability to define concentration differences to the precision of his spectrophotometer; this is in contrast to use of a chromophore of high sensitivity which, given the same degree of spectrophotometric precision, expresses itself as a smaller and thus more accurately defined concentration difference.
• the present invention accomplishes a fully enzymatic assay for triglyceride without use of a protease and with the novel inclusion of iron in the reaction medium, while avoiding various disadvantages of the prior art, and of earlier prior art attempts at improvement.
• the procedure of this invention is capable of being performed rapidly and in a single tube, thus representing great advantages in that larger numbers of assays may be performed by a single technician in a reasonable time frame.
• the method of this invention is free from the more common sources of interferences in triglyceride assays, such as that usually encountered from phospholipids and glucose. Freedom from phospholipids is achieved by the use of the enzyme lipase which is highly specific for triesters of glycerol; glucose is not a source of interference, due to the mild reaction conditions employed in the assay system.
• the method of the present invention does not require expensive instrumentation or expensive equipment capable of measurements in the U.V. Range for accomplishment of the assay.
• the present invention for the first time in triglycerides assays, achieves an accuracy sensitivity, and reliability, with health-improving and life-saving benefits which the prior art assays have hoped for but failed to achieve; for although prior art methods have overcome certain disadvantages of prior assays, none has been successful in avoiding other disadvantages or in accomplishing the benefits and advantages of this present invention.
• the reaction of the present invention utilizes an enzymatically catalyzed hydrolysis with lipase.
• the glycerol thus liberated is then converted to dihydroxy acetone and NADH through the following course of reactions as suggested by several authors: 9 , 10
• ATP Addenosine Triphosphate
• ADP Adenosine Diphosphate
• NAD Nicotinamide Adenine Dinucleotide
• NADH Nicotinamide Adenine Dinucleotide (reduced);
• AMB-610 9-(2-pyridyl)-acenaphtho[1,2-e]-as-triazine sulfonate
• the increase in sensitivity is about six-fold, in comparison to the cited Bucolo and David alternative.
• the interference from lipemic specimens is greatly reduced over those methods which require measurement at lower wavelengths such as 340 nm.
• a ferrous indicator such as 9-(2-pyridyl)-acenaphtho[1,2-e]-as-triazine sulfonate
• a serum sample containing triglyceride is then added to the above solution, along with a solution containing an electron transfer agent (such as phenazine methosulfate), nicotinamide adenine dinucleotide (oxidized), and ferric ions.
• an electron transfer agent such as phenazine methosulfate
• nicotinamide adenine dinucleotide oxidized
• ferric ions such as phenazine methosulfate
• the mixture is then incubated at a temperature of 37° C. for a period of time, and the resulting blue color representing triglyceride is then read in a spectrophotometer at a wavelength of 610 nanometers; and the resulting absorbance is recorded and is used to calculate the triglyceride content of the original sample by comparison to the absorbance of a standard solution of triglyceride which has been treated in an identical manner as the serum sample.
• the resultant solution is then mixed thoroughly and incubated at a temperature of 37° C. for 20 minutes.
• the solution containing phenazine methosulfate it is preferable that the solution be protected from the presence of strong light or sunlight.
• the absorbance of the mixture is then measured in a spectrophotometer at 610 nanometers against a reagent blank which is prepared in an identical manner as the serum reaction mixture, except that 20 microliters of distilled water is substituted for the serum sample.
• the triglyceride content of the serum sample is then calculated by comparison of the measured absorbance to the absorbance developed by a solution of known triglyceride content which has been treated in an identical manner as the serum sample.
• a 7 microliter serum sample is dispensed into a reaction vessel; and to this vessel are simultaneously added a 0.5 milliliter aliquot of a reagent containing 36 mmol/L of adenosine triphosphate, 40.9 mmol/L MgSO 4 , 1,318,000 units/L of lipase, 6,040 I.U./L of glycerol-3-phosphate dehydrogenase, and 570 I.U./L of glycerol kinase; a 1.5 ml aliquot of a reagent containing 47 ⁇ mol/L of phenazine methosulfate, 0.466 mmol/L ferric ions and 27.3 mmol of nicotinamide adenine dinucleotide (oxidized); and a 1.5 ml aliquot of a 0.17 molar triethanolamine buffer, pH 9.4, containing 2.1 m
• the solution is then mixed and incubated at a temperature of 37° C. for approximately 15 minutes.
• the final absorbance of the mixture is then spectrophotometrically measured at 610 nanometers, and the amount of triglyceride in the original sample is calculated as in Example 1 above. Under these conditions a linear absorbance response has been demonstrated in serum samples containing up to 1000 mg/dl of triglyceride.
• Example 1 As in Example 1 except that 1.6 mmol/L of bathophenanthroline sulfate is substituted for the 1.6 mmol/L of 9-(2-pyridyl)-acenaphtho[1,2-e]-as-triazine sulfonate.
• the other iron chelators such as bathophenanthroline sulfonate and FerroZine 13
• the other iron chelators such as bathophenanthroline sulfonate and FerroZine 13
• the other iron chelators may be substituted on an equimolar basis for the 9-(2-pyridyl)-acenaphthol[1,2-e]-as-triazine sulfonate set forth in Example 2.
• the exact unitage of lipase may be lowered with an accompanying increase in incubation time to effect complete hydrolysis.
• composition of the buffer which has been optimized in the disclosed assay systems, may be modified slightly as to type the pH without serious effects on the assay system.

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Cited By (33)

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• 1977
  • 1977-12-07 US US05/858,187 patent/US4245041A/en not_active Expired - Lifetime
• 1978
  • 1978-08-07 JP JP9605178A patent/JPS5480192A/ja active Pending
  • 1978-10-02 CA CA312,452A patent/CA1125151A/en not_active Expired
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  • 1978-11-16 FR FR7832374A patent/FR2411411A1/fr active Granted
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